The aims of this proposal are to define the basis of imidazole action against drug-resistant strains of Plasmodium falciparum, and to determine whether the imidazoles enhance the antimalarial activity of other agents against the parasite in vitro and in vivo. We will define the extent of the sterol pathway in P. falciparum, and the effect of the imidazoles on it to determine whether the imidazoles inhibit this pathway in plasmodia, as they do in fungi. Based on our recent studies, we will use mevinolin with radiolabelled mevalonate (to enhance the specific activity of the compounds produced from this pathway), and will identify the labelled components by HPLC and TLC examination of fractions derived from the Bligh-Dyer extraction of parasitized red cells. We will use a similar strategy (mevinolin + radiolabelled isopentenyl adenine) to define the effect of this pathway on parasite nucleic acid synthesis. Because imidazoles inhibit the antoxidant (peroxidase and catalase) activity of fungi, and because we have recently shown that inhibitors of these enzymes prevent parasite growth, we will determine whether the imidazoles act against plasmodia by inhibiting these enzymes, or by increasing the concentrations of the toxic oxygen metabolites they regulate. We will measure the direct effects of the imidazoles on these enzymes using spectrophotometric assays of enzyme activity, and their effects on the concentrations of superoxide, hydrogen peroxide, and hydroxyl radical by the reduction of ferricytochrome c, the oxidation of leukodiacetyl-2,7-dichlorofluorescein, ethylene production from Alpha-keto-Gamma-thiomethyl butyric acid, and the detection of free radicals using electron spin resonance and the spin trap DMPO. We will define the antimalarial activity of these compounds (e.g., mevinolin, and the inhibitors of antoxidant enzymes) in combination with the imidazoles both in vitro (using 3H-hypoxanthine uptake) and in vivo (with the murine-P. berghei model). The results of these studies should facilitate the development of more effective chemotherapy against multiply-resistant strains of P. falciparum, and better define the biology of the parasite and the red cell-parasite relationship (by defining the dependence of the parasite on the antoxidant enzymes and sterols of the red cell).